With the upsurge in antibiotic resistance among pathogens and the increase in hospital acquired infections such as MRSA and C. difficile there is a renewed urgency in discovering novel antimicrobial compounds to combat these diseases. First described in 1935, C. difficile was not recognised as the causative agent of nosocomial diarrhoea until the 1970s (George et al., 1978; Hall & O'Toole, 1935). However, Clostridium difficile associated disease (CDAD) is the now most common hospital acquired diarrhoea and is a major problem of gastroenteritis infection and antibiotic associated diarrhoea in nursing homes and care facilities for the elderly. Indeed, the health protection agency in UK reported 32,189 cases of CDAD for the first 6 months of 2007 in UK. The main predisposing factor for the acquisition of CDAD is antibiotic therapy. In the 1970s the administration of clindamycin followed by ampicillin and amoxicillin were implicated as the inducing agents of CDAD; these were replaced by cephalosporins in the 1980s and more recently by flouroquinolones (Aronsson et al., 1985; Bartlett, 2006; Winstrom et al., 2001). There is also the added problem of the hyper-virulent strain of C. difficile PCR ribotype 027, the incidence of which is increasing in US, Canada and Europe (Bartlett, 2006). Antimicrobial peptides produced by bacteria, now designated as bacteriocins, first came to prominence ˜80 years ago with the discovery by Rogers & Whittier (1928) of nisin by Lactococcus lactis subsp. lactis which demonstrated a broad spectrum of activity against other lactic acid bacteria (LAB) and other Gram positive organisms. While the bacteriocins produced by LAB are the most widely studied and tend in the main to have a broad spectrum of activity, antimicrobial compounds are produced by many other bacterial species including Gram positive organisms Bacillus (Ahern et al., 2003; Bizani et al., 2005; Cherif et al., 2003; Cherif et al., 2001; Seibi et al. 2007, Teo & Tan, 2005); Clostridium (Kemperman et al., 2003), Gram negative organisms E. coli (Trautner et al., 2005), Shigella (Padilla et al., 2006).
Work carried out previously on various strains of B. thuringiensis have yielded a variety of bacteriocins (Ahern et al. 2003, Chehimi et al. 2007, Cherif et al. 2001, Favret and Yousten 1989, Gray et al. 2006a, Gray et al. 2006b,) demonstrating bactericidal properties against B. thuringiensis strains, B. cereus strains, and Listeria monocytogenes strains. However, these bacteriocins do not exhibit two-component activity.
Previous work by Yudina et al. describes proteins of parasporal crystals (Cry proteins) from entomopathogenic bacterium B. thuringiensis (subsp. Kurstaki, galleriae, tenebriois) as well as some fragments thereof, obtained by limited proteolysis which are capable of antimicrobial action against anaerobic bacteria and C. butyricum, C. acetobutylicum and Methanosarcina barkeri. U.S. Pat. No. 7,247,299 describes antimicrobial heat-stable compounds isolated from a novel strain of B. subtilis (deposited 8.5.05) isolated from the GIT of poultry, which are effective against C. perfringens, C. difficile, Campylobacter jejuni, Camp. coli, and S. pneumoniae. U.S. Pat. No. 7,144,858 describes the synthesis of new antibiotic compounds for use against Gram positive bacteria such as Bacillus (including B. thuringiensis), Clostridium (including C. difficile), Streptococcus, Mycobacterium, and Staphylococcus. US Application 20080213430 describes the artificial synthesis and recombinant expression of antibacterial peptides against bacteria such as B. subtilis, C. difficile, E. coli, Staphylococcus, and the like. However, these peptides have a broad spectrum of inhibition against a wide range of Gram positive organisms. Previous work using the naturally occurring lantibiotics lacticin and nisin have shown that these microbially derived peptides are effective in killing C. difficile at concentrations that compare well with commonly used antibiotics such as vancomycin and metronidazole (Bartoloni et al., 2004; Rea et al., 2007).
However, these lantibiotics have a broad spectrum of inhibition against a wide range of Gram positive organisms including those which would be considered beneficial to human gut health such as Lactobacillus and Bifidobacterium. Indeed, previous work in this laboratory has demonstrated that lacticin 3147 negatively affects the levels of Lactobacillus and Bifidobacterium in faecal fermentation (Rea et al., 2007). The aim of this study was therefore to isolate bacteria which produce narrow spectrum antimicrobial compounds which target C. difficile. To this end spore forming bacteria in the human gut were targeted; this would not be an obvious source of antimicrobials against C. difficile. 